BACKGROUND: Collagen XI is a key structural component of the extracellular matrix and consists of three alpha chains. One of these chains, the alpha1 (XI), is encoded by the COL11A1 gene and is transcribed to four different variants at least (A, B, C and E) that differ in the propensity to N-terminal domain proteolysis and potentially in the way the extracellular matrix is arranged. This could affect the ability of tumor cells to invade the remodeled stroma and metastasize. No study in the literature has so far investigated the expression of these four variants in breast cancer nor does a method for their accurate quantitative detection exist. METHODS: We developed a conventional PCR for the general detection of the general COL11A1 transcript and real-time qPCR methodologies with dual hybridization probes in the LightCycler platform for the quantitative determination of the variants. Data from 90 breast cancer tissues with known histopathological features were collected. RESULTS: The general COL11A1 transcript was detected in all samples. The developed methodologies for each variant were rapid as well as reproducible, sensitive and specific. Variant A was detected in 30 samples (33 %) and variant E in 62 samples (69 %). Variants B and C were not detected at all. A statistically significant correlation was observed between the presence of variant E and lymph nodes involvement (p = 0.037) and metastasis (p = 0.041). CONCLUSIONS: With the newly developed tools, the possibility of inclusion of COL11A1 variants as prognostic biomarkers in emerging multiparameter technologies examining tissue RNA expression should be further explored

OBJECTIVES: Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for acute kidney injury. NGAL can be measured in both blood and urine. Apart from kidney injury, NGAL levels in both plasma and urine can be influenced by various pathological situations. Accurate evaluation and comparison of results deriving from clinical studies require robust assays, appropriate specimen handling and reference intervals that will reflect its levels in a healthy population for both biological matrices. METHODS: We report the analytical validation of a latex particle-enhanced turbidimetric immunoassay (PETIA) aimed to measure NGAL in plasma and urine on an automated biochemistry analyzer (ABBOTT-Architect-8000). Assay performance characteristics were evaluated using standard protocols. Urine and plasma specimen storage requirements were determined and reference ranges for blood and urine were determined using healthy controls. RESULTS: The assay is precise (total CV%<4.8%), and sensitive (limit of quantification: 8.4 ng/mL for plasma and 9.0 ng/mL for urine), showing no hook effect. Calibration is stable for at least 30 days. The assay showed excellent linearity over the studied interval (20-4450 ng/mL). The analyte is stable at 4 degrees C for at least 5 days, and at 20 degrees C for 4h. Gender specific reference ranges for plasma (male: 38.7-157.6 ng/mL, female: 24.4-142.5 ng/mL) and unisex for urine (<9.0-49.41 ng/mL) are proposed. CONCLUSION: Our data indicate that NGAL can be measured with adequate precision and sensitivity on automated biochemistry analyzers and its measurement could easily be added to a standard panel to screen kidney diseases

The recent revision of ISO15189 has further strengthened its position as the standard for accreditation for medical laboratories. Both for laboratories and their customers it is important that the scope of such accreditation is clear. Therefore the European co-operation for accreditation (EA) demands that the national bodies responsible for accreditation describe the scope of every laboratory accreditation in a way that leaves no room for doubt about the range of competence of the particular laboratories. According to EA recommendations scopes may be fixed, mentioning every single test that is part of the accreditation, or flexible, mentioning all combinations of medical field, examination type and materials for which the laboratory is competent. Up to now national accreditation bodies perpetuate use of fixed scopes, partly by inertia, partly out of fear that a too flexible scope may lead to over-valuation of the competence of laboratories, most countries only use fixed scopes. The EA however promotes use of flexible scopes, since this allows for more readily innovation, which contributes to quality in laboratory medicine. In this position paper, the Working Group Accreditation and ISO/CEN Standards belonging to the Quality and Regulation Committee of the EFLM recommends using an approach that has led to successful introduction of the flexible scope for ISO15189 accreditation as intended in EA-4/17 in The Netherlands. The approach is risk-based, discipline and competence-based, and focuses on defining a uniform terminology transferable across the borders of scientific disciplines, laboratories and countries

BACKGROUND: Age-related macular degeneration (AMD) is a degenerative ocular disease, which may lead to loss of central vision. In Caucasian populations, a strong correlation has been established with polymorphism Y402H (rs1061170) in the complement factor H gene (CFH). The H131R polymorphism (rs1801274) in the FCGR2A gene has been associated with many inflammatory diseases, but has not been investigated in relation to AMD. The goal of our study was the development of a novel method for Y402H (g.43097C>T) genotyping, the confirmation of its association with AMD in the Greek population and the investigation of the H131R polymorphism in AMD. METHODS: DNAs were extracted from blood samples of 120 patients with the severe wet form of AMD and 103 age- and sex-matched controls, all of whom were clinically evaluated. A real-time PCR and melting curve analysis method for Y402H genotyping was developed in the LightCycler platform, after in silico design of appropriate primers and probes. Genotyping for H131R was performed using a real-time PCR method previously described by our group. RESULTS: The novel genotyping method for Y402H in the CFH gene is fast, reproducible (Efficiency=1.79, reproducibility CVCq=3.33%, Tm C allele 53.36 degrees C and T allele 61.91 degrees C, DeltaTm=8.55) and accurate as results were confirmed with the gold standard DNA Sequencing method. CONCLUSIONS: The present study confirmed the association between CFH Y402H SNP and wet AMD in the Greek population (OR=1.77, p=0.002). FCGR2A H131R polymorphism was investigated for the first time in this present study for possible correlation with wet AMD and a statistically significant association was detected (OR=1.74, p=0.006), that awaits further confirmation in a larger set of samples

Oxidative stress refers to cellular or molecular damage caused by reactive oxygen species, which especially occurs in age-related conditions as a result of an imbalance between the production of reactive oxygen species and the antioxidant defense response. Dry age-related macular degeneration (AMD) and exfoliation syndrome (XFS) are two common and complex age-related conditions that can cause irreversible vision loss. Two subtypes of AMD, which is the leading cause of blindness in the Western world, exist: the most prevalent dry type and the most severe wet type. Early dry AMD is characterized by formation of drusen, which are sub-retinal deposits, in the macular area and may progress to geographic atrophy with more dramatic manifestation. XFS is a systemic disorder of the extracellular matrix characterized by the accumulation of elastic fibrils that leads, in most cases, to glaucoma development with progressive and irreversible vision loss. Due to the aging population, the prevalence of these already-widespread conditions is increasing and is resulting in significant economic and psychological costs for individuals and for society. The exact composition of the abnormal drusen and XFS material as well as the mechanisms responsible for their production and accumulation still remain elusive, and consequently treatment for both diseases is lacking. However, recent epidemiologic, genetic and molecular studies support a major role for oxidative stress in both dry AMD and XFS development. Understanding the early molecular events in their pathogenesis and the exact role of oxidative stress may provide novel opportunities for therapeutic intervention for the prevention of progression to advanced disease